Vectors

ABSTRACT

The present invention relates to a transfer vector for inserting a gene into a genetic locus of a baculovirus sequence. The transfer vector comprises an expression cassette comprising a eukaryotic promoter operably linked to the gene and a bipartite selection cassette. The present invention also relates to methods of using the transfer vector and derived bacmids and baculoviruses.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 13/128,629 filed Jul.21, 2011, U.S. Pat. No. 9,212,374, which is the U.S. National Stage ofPCT/GB2009/002647 filed Nov. 11, 2009, which claims priority to GB0820631.1 filed Nov. 11, 2008.

FIELD

The present invention relates to a transfer vector for inserting a geneinto a genetic locus of a baculovirus sequence. The present inventionalso relates to methods of using the transfer vector and derived bacmidsand baculoviruses.

BACKGROUND

The baculovirus expression system has been used to express manythousands of proteins in eukaryotic cells for structural and biochemicalstudies (14). In addition to its ability to express single recombinantproteins the baculovirus system has also been used to co-expressmultiple proteins that form complexes (15-22). This is important asgenome-wide interaction screens are revealing that many criticalfunctions of living cells are carried out by multi-subunit proteincomplexes (23). Two main strategies have been used to achieveco-expression of proteins using the baculovirus system. The coinfectionof cells with two or more viruses each of which expresses onerecombinant protein is by far the most common approach (19,24,25).However, often yields of protein complexes from these studies are veryvariable and co-infection of the same cells by two differentbaculoviruses does not follow the poisson distribution (26). Thus whilethe co-infection approach is technically straightforward, it ispractically limited to recovery of relatively simple complexes forapplications that do not require large amounts of purified proteins.

For applications such as structural studies and large vaccine trialswhere more protein is required, and for more complicated complexesformed from many subunits, the alternative approach of co-expressingproteins from multiple similar expression cassettes inserted at thepolyhedrin or P10 loci has been used (15-18,22,27). This approach hasthe advantage that every cell in the culture that is infected withrecombinant virus expresses all of the proteins required for formationof the protein complex in a reproducible manner. Where the coinfectionand single baculovirus approaches have been compared, the latter hasdemonstrated significantly better yield of recombinant complex (28).However, expression of multiple proteins using a single recombinantvirus is not without drawbacks. Although rod shaped baculovirus appearsquite tolerant of insertions in its genome, genes are transferred to thevirus by homologous recombination and the transfer vector must be of asize that is easily manipulated in E. coli. Therefore there is a limitto the number of genes that can be inserted into a transfer vector. Inpractice this means that it is rarely possible to express more than fourproteins from a single locus. In addition, baculoviruses containsequences and express proteins that promote homologous recombination(29-32). Therefore viruses that contain large amounts of repeatedsequence are prone to rearrangement and recombination (21,33,34).

The single baculovirus coexpression approach has been modified byinsertion of a loxP site at the chiA/cathepsin locus of a bacmid thatalready contains the Tn7 target at the polyhedrin locus (20). Thisallows insertion of multiple expression cassettes at each of these lociusing recombination in E. coli. As might be expected given that thissystem relies on the iterative duplication of an expression cassettethat has been modified to express different genes there is evidence thatinserts into this system are somewhat unstable genetically (21).

Recently baculovirus research has benefited from use of the ETrecombination system which has allowed selective knockout of viral genes(35-49). However, the potential for this technique to engineer andfacilitate the expression of proteins at genetic loci other than p10 andpolyhedrin has not been examined.

The present invention relates to a method for the efficient expressionof multiple recombinant proteins (i.e., non-baculoviral proteins) from asingle baculovirus genome. The method allows single protein expressioncassettes to be inserted efficiently at different loci within the viralgenome using the ET recombination system. Furthermore, the method allowsfor the expression of complexes with numerous subunits.

The expression of single proteins using baculovirus as an expressionsystem is well established. The basic methodology of the expressionsystem has changed very little since its first production. At 133 kb thebaculovirus dsDNA genome is too large to manipulate directly. Genesencoding foreign proteins are therefore cloned, in E. coli, into vectorscontaining an expression cassette from the AcMNPV insect virus and thisis then introduced with viral DNA into insect cells where viral andcellular proteins mediate homologous recombination that results in theproduction of an infectious (with respect to insect cells) virus thatexpresses the foreign protein. Some variations on this theme havealready been generated, including viral genomes that only replicate ifrecombination occurs (1,2), and performing recombination in E. coli witha bacmid based on the use of transposon Tn7 (3) to speed up theselection of recombinant virus. The expression of multi-proteincomplexes in insect cells has also been achieved using the baculovirusexpression system as briefly discussed above. Initially, multipleviruses each expressing a single protein were used to co-infectsusceptible cells and the protein complex purified after expression.However, this is at best inefficient and not reproducible enough to befeasible for scale-up. As an alternative, vectors that incorporatemultiple expression units but recombine with a single virus geneticlocus were produced (4-6). These vectors had the advantage that theyproduced all recombinant protein subunits in the every infected cell andresulted in significantly higher yields of recombinant protein complex.In addition, because only a single virus expresses all the proteins theresults of infections were much more reproducible. The disadvantage ofthese vectors is twofold. Firstly, because the vectors contain repeatedsequences and the baculovirus expresses proteins that promote homologousrecombination, expression, particularly from constructs containing threeor four expression units, is not always very stable genetically throughlarge numbers of viral passages (which are necessary during industrialscale-up for baculovirus protein expression). Secondly, there is apractical limit to the size of insert that can be easily maintained andmanipulated in E. coli which limits the number of proteins that can beexpressed from these vectors. Another approach that has been proposedfor the production of multiple proteins is the insertion of foreignprotein(s) at one genetic locus in baculovirus, then selection of virusexpressing that protein and the use of this viral genome to recombine ata second genetic locus to express other proteins (7). However, becauseof the high degree of technical skill and amount of time involved inthis approach (16 days for the first locus and 25 days for eachsubsequent locus) this approach has hardly ever been used. A recentdevelopment was to modify the BAC based Tn7 transposon system to inserta LoxP site at a second genetic locus in baculovirus to also allow theinsertion of extra genes at this locus in E. coli (8). However, thismethod still suffers from the problems that repeat sequences fromduplication of promoters in the bacterial vectors will mean that theconstruct is genetically unstable for multiple viral passages andtransfer vectors will quickly reach the maximum practical size formanipulation in E. coli.

The method of the present invention overcomes at least some of theproblems inherent in the prior methods, such as genetic instability andthe requirement for multiple rounds of recombination.

SUMMARY

According to a first aspect, the present invention provides a transfervector for inserting a gene into a genetic locus of a baculovirussequence comprising:

an expression cassette comprising a eukaryotic promoter operably linkedto the gene;

a bipartite selection cassette comprising:

-   -   (i) an expressible sequence encoding a first selectable marker;        and    -   (ii) an expressible sequence encoding a second selectable        marker; and

sequences flanking the expression and bipartite selection cassettes,wherein the sequences substantially correspond to sequences of thegenetic locus within the baculovirus sequence.

The transfer vector of the present invention can be used to efficientlyinsert a gene into a genetic locus of a baculovirus sequence and toselect for the baculovirus sequences that comprise the gene. Inparticular, it has been found that by using a bipartite selectioncassette a substantial increase in identification of positive clones(i.e., baculovirus sequences containing the inserted gene) was obtainedcompared to selection using a single selection marker. This provides asignificant advantage over the use of a single selection marker. Only asmall number of baculoviruses are transformed and hence the marker isonly expressed at very low levels. For example, when the marker isantibiotic resistant only very low levels of antibiotic can be used.This results in the selection of bacterial variants that are resistantto the antibiotic used. Hence some bacteria growing on the plates maynot contain the modification. Using two positive selection methodsovercomes the possibility of such false positive selections.

The transfer vector of the present invention can be any suitable vector,provided it is capable of recombining with a baculovirus sequence viahomologous recombination to insert a gene into the baculovirus sequence.Suitable transfer vectors are well known to those skilled in the art.Key features of the transfer vector are discussed further below.

The gene to be inserted into the baculovirus sequence can be anyheterologous gene (i.e., non-baculoviral gene). It is preferred that theheterologous gene encodes a subunit of a protein complex. Theheterologous gene may encode a viral protein, a component of a receptoror a chaperone complex. It is particularly preferred that theheterologous gene encodes a viral protein that together with other viralproteins can form a virus like particle (VLP).

The genetic locus at which the gene is inserted into the baculovirussequence can be any suitable locus that allows expression of theinserted gene and also does not prevent the ability of the baculovirussequence from replicating. Further, the locus used should not affect theability of baculovirus to infect cells, i.e. replicate or spread fromcell to cell. Such loci include the polyhedrin and the p10 loci. Furtheradditional suitable loci identified by the present inventors includectx, egt, 39k, orf51, gp37, iap2 and odv-e56.

The baculovirus sequence can be any suitable baculovirus sequence thatcan replicate in an insect cell and a prokaryotic cell such as E. coli.In particular, any viral baculovirus genome that contains a BAC repliconmay be used. Suitable baculovirus sequences include the AcMNPV bacmid.

The term “expression cassette” refers to the combination of elementsrequired for expression of the gene. Accordingly, the expressioncassette comprises a suitable promoter allowing expression of theencoded gene in eukaryotic cells (i.e., insect cells), and a suitablepolyadenylation signal sequence flanking the gene sequence. Expressioncassettes for the expression of genes in eukaryotic cells are well knownto those skilled in the art. Particularly preferred promoters includethe baculovirus viral late promoters (e.g. p35) or viral very latepromoters (e.g. polyhedrin and p10 promoters). Suitable polyadenylationsignal sequences include the tryptophan hydroxylase (Tph)polyadenylation sequence, or polyadenylation sequences from baculovirusgenes.

The expression cassette can comprise more than one gene (e.g., 2, 3 or 4genes) resulting in the expression of more than one gene. However,generally, it is preferred that the expression cassette comprises asingle gene.

The bipartite selection cassette has been found to improve theidentification and isolation of clones that have successfully receivedthe gene. The bipartite selection cassette comprises 2 differentselectable markers. The cassette also comprises any elements requiredfor expression of the markers in a bacterial cell such as one or morebacterial promoters. The step of selecting sequences that have receivedthe gene comprises selecting for sequences that comprise both theselectable markers. This selection step is carried out in bacterialcells, e.g., E. coli. The selectable markers may be any markers thatallow the cells comprising the cassette to be selected. For example, themarkers may be visible such as a marker that causes the appearance orchange of colour of a cell or colony. Alternatively, the marker mayconfer resistance to, for example, an antibiotic. The two selectablemarkers may be the same type of marker, e.g. resistance to two differentantibiotics, or different types of marker, e.g. antibiotic resistanceand a visual marker. The first selectable marker is preferably a visualmarker, such as the LacZalpha fragment, which allows cells expressingthe LacZalpha fragment to appear blue in the presence of IPTG and X-gal.The second selectable marker is preferably any marker that confersantibiotic resistance, such as resistance to chloramphenicol,tetracycline, puromycin, ampicillin, penicillin, apramycin, kanamycin orphleomycin. It is particularly preferred that the second marker confersresistance to phleomycin.

In a preferred embodiment the bipartite selection cassette is flanked byLoxP recombination sequences. LoxP recombination sequences combinetogether in the presence of Cre recombinase resulting in the deletion ofthe intervening sequence. Accordingly, the bipartite selection cassettecan be removed enabling the same selectable markers to be used wheninserting any additional genes into the baculovirus sequence. Preferablythe LoxP sites are modified to ensure that only a single round ofrecombination can occur between the sites. Suitable modified LoxP sitesare known to those skilled in the art, for example, the Lox66 and Lox71modified sites.

The sequences flanking the expression and bipartite selection cassettessubstantially corresponds with sequences of the genetic locus allowinghomologous recombination to occur between the transfer vector and thebaculovirus sequence. Each flanking sequence is preferably at least 20bp in length, more preferably at least 30 bp in length and even morepreferably at least 50 bp in length, and has a sequence allowingspecific recombination with a sequence of the genetic locus.

According to a second aspect, the present invention provides a methodfor producing a recombinant bacmid comprising:

bringing a bacmid and the transfer vector according to the first aspectof the present invention together to allow homologous recombination; and

selecting for a recombinant bacmid that comprises the bipartiteselection cassette.

The recombinant bacmid can be selected using standard techniquesdepending on the bipartite selection cassette.

The method for producing a recombinant bacmid may additionally comprisedetecting the presence of the gene in the bacmid or its expressionproduct. Suitable screening techniques can be used depending on thespecific gene.

Furthermore, when the selection cassette is flanked by LoxPrecombination sequences, the method preferably further comprisesinducing recombination between the LoxP sites, e.g., by exposing thebacmid to Cre recombinase, to remove the bipartite selection cassette.The Cre recombinase is preferably under the control of an induciblepromoter, such as the arabinose promoter. The advantage of removing thebipartite selection cassette is that a further transfer vector can berecombined with the bacmid, wherein the further transfer vector containsthe same bipartite selection cassette. Selection for the furtherrecombined bacmid can be performed using the same techniques as usedfollowing the first round of recombination.

Generally, the method of producing a recombinant bacmid is performed ina prokaryotic cell as a prokaryotic cell based system is easier andquicker to manipulate. It is preferred that the prokaryotic cell basedsystem is conducive to homologous recombination. Such systems includethe lambda red recombination system, which is described in Europeanpatent application EP-A-1291420. Preferably, the method of producing therecombinant bacmid is performed in E. coli. It is particularly preferredthat the method is performed using E. coli cell line EL350, which hasboth an integrated lambda prophage expressing exo, bet and gam undercontrol of the temperature regulated lambdaPL promoter, and Crerecombinase under control of an arabinose inducible promoter.

The method of producing the recombinant bacmid can be repeated a numberof times so that the bacmid can contain a number of heterologous genes,i.e., 1, 2, 3, 4, 5, 6, 7, 8 or more heterologous genes. By endeavoringto use different promoters for expressing each inserted gene and as eachgene is inserted at a different genetic locus, it is possible to reduceor avoid having repeated sequences in the bacmid and thereby ensure thatthe bacmid has good genetic stability. It is further preferred that atleast one essential gene is positioned between each insertedheterologous gene. Such an arrangement ensures that if homologousrecombination occurs between any of the inserted sequences, an essentialgene will be deleted resulting in a non-viable bacmid.

According to a third aspect, the present invention provides arecombinant bacmid obtained by the method according to the second aspectof the present invention. The bacmid comprises the inserted gene and mayalso comprise remnants of the LoxP sequences. These remnants may be usedas markers to identify the bacmids. The bacmid preferably comprises atleast 6, 7, 8 or 9 heterologous genes.

According to a fourth aspect, the present invention also provides amethod for producing a recombinant baculovirus comprising culturing aeukaryotic cell containing the recombinant bacmid obtained by the methodaccording to the second aspect of the present invention under conditionsso that a baculovirus is produced.

Preferably the eukaryotic cells are insect cell, more preferably derivedfrom the insects Spodoptera frugiperda or Trichoplusia ni e.g. (Sf21, SPor TN 5B-1-4) insect cells. The baculovirus can be isolated usingstandard techniques and the expression of the inserted heterologous genetested.

According to a fifth aspect, the present invention also provides arecombinant baculovirus obtained by the method according to the fourthaspect of the present invention.

According to a sixth aspect, the present invention also provides arecombinant bacmid or a recombinant baculovirus that expresses aplurality of heterolgous proteins, wherein each protein is expressedfrom a separate genetic locus of the bacmid or baculovirus. It isfurther preferred that at least one essential gene is positioned betweeneach genetic locus expressing a heterologous protein. Such anarrangement ensures that if homologous recombination occurs betweengenetic loci expressing a heterologous gene an essential gene is deletedresulting in a non-viable bacmid or baculovirus. Preferably the bacmidor baculovirus expresses at least 3, more preferably at least 5 and mostpreferably at least 8 proteins. The proteins are heterolgous, i.e., arenot encoded by naturally occurring baculoviruses. It is furtherpreferred that the encoded heterologous proteins are subunits of aprotein complex, such as a VLP, a receptor complex or a chaperonecomplex.

It is further preferred that the separate genetic loci of therecombinant bacmid or baculovirus are selected from ctx, egt, 39k,orf51, gp37, iap2, odv-e56 and p10. The position of such loci arespecifically described with reference to baculovirus AcMNPV in Table 1below. One skilled in the art can easily determine the location of theloci in other baculovirus strains and bacmids using this information.

TABLE 1 The genetic loci referred to above are defined below withreference to the reference accession sequence for AcMNPV (accessionnumber NC001623). Locus boundaries ctx 2028-2296 egt 11310-13091 39k29196-30070 orf51 43154-44337 gp37 52192-52327 iap2 60983-61823 p35116282-117460 p10 118767-119135 odv-e56 128947-130166

According to a seventh aspect, the present invention also provides arecombinant bacmid or a recombinant baculovirus wherein an expressioncassette encoding a heterologous protein is inserted at one or more ofthe following genetic loci: ctx, egt, 39k, orf51, gp37, iap2 andodv-e56.

It has been found that these genetic loci specifically allow high levelexpression of the heterologous protein without disrupting the essentialfunctions of the bacmid or baculovirus.

Preferably the recombinant bacmid or recombinant baculovirus encodes aplurality of proteins, each expression cassette being inserted at adifferent genetic locus. It is further preferred that the recombinantbacmid or recombinant baculovirus encodes at least 3, more preferably atleast 5 and most preferably at least 8 proteins. It is further preferredthat the encoded heterologous proteins are subunits of a proteincomplex, such as a VLP, a receptor complex or a chaperone complex.

According to an eighth aspect, the present invention also provides atransfer vector for inserting a gene into a genetic locus of abaculovirus sequence comprising:

an expression cassette comprising a eukaryotic promoter operably linkedto the gene; and

sequences flanking the expression cassette, wherein the sequencessubstantially correspond to sequences of the genetic locus within thebaculovirus sequence, wherein the genetic locus is selected from: ctx,egt, 39k, orf51, gp37, iap2 and odv-e56.

As indicated above, it has been found that by inserting the gene intothe recited genetic loci high level expression of the gene can beobtained without disrupting the essential functions of the baculovirussequence.

According to a ninth aspect, the present invention also provides amethod for producing a recombinant bacmid comprising:

bringing a bacmid and the transfer vector according to the eighth aspectof the present invention together to allow homologous recombination; and

selecting for a recombinant bacmid that comprises the expressioncassette.

Methods for producing such a recombinant bacmid and selecting for such abacmid are well known to those skilled in the art. The present inventionalso provides a recombinant bacmid obtained by this method.

According to a tenth aspect, the present invention also provides amethod for producing a recombinant baculovirus comprising producing arecombinant bacmid by the method of the present invention and culturinga eukaryotic cell containing the bacmid so that a baculovirus isproduced.

Methods for producing such a recombinant baculovirus are well known tothose skilled in the art. The present invention also provides arecombinant baculovirus obtained by this method.

The present invention also provides a cell containing a transfer vector,a bacmid, or a baculovirus according to any aspect of the presentinvention.

The present invention also provides a method for producing one or moreproteins comprising culturing the recombinant bacmid or baculovirusaccording to any aspect of the present invention under suitableconditions. The one or more proteins are encoded by the one or moregenes and may combine to form a protein complex such as a VLP or achaperone complex. The one or more proteins can be isolated usingstandard techniques well known to those skilled in the art.

BRIEF SUMMARY OF THE DRAWINGS

The present invention is now described by way of example only withreference to the following figures.

FIG. 1A shows an improved selection of recombinants from ET cloning.Cartoon of DNA used for cat and bipartite recombinations. Bothconstructs had the same baculovirus flanking sequences (AcMNPV), Renillaluciferase expression cassette (Pp35-Rluc-Tph) and loxP sites (LoxP).The bipartite selectable marker also had zeocin resistance gene (ZeoR)and LacZα fragment. In the chloramphenicol resistant construct this wasreplaced with the cat gene.

FIG. 1B is a bar graph showing the number of positive bacterial coloniesfollowing ET recombination. DNA used for recombination was producedeither by PCR or restriction enzyme digest, as indicated.

FIG. 1C is a digital image showing the correct insertion of Rlucexpression cassette confirmed by PCR using one primer in the transferredDNA and one flanking the target locus in the baculovirus DNA. Thecorrect PCR product (arrowed) would only be produced following correctintegration. Results for 12 independent recombinants (1-12), no templatePCR control (13), unmodified bacmid template (14) and transfer DNAtemplate (15) are shown. Lane M is marker DNA.

FIG. 1D is a bar graph showing renilla luciferase activity (relativelight units) at 48 hours post infection in cell lysate from cellsinfected with passage 2 of recombinant bacmids 1-12 from C. Backgroundactivity in equivalent lysates from uninfected (cells) and unmodifiedbacmid (AcMNPV) infected cells were more than 106 fold lower.

FIG. 2A shows the selective removal of marker genes. Cartoon showingstrategy for ET recombination of an expression cassette (expression)into the AcMNPV Bacmid DNA and selective removal of only the markergenes (selection) by Cre mediated recombination.

FIG. 2B shows the selective removal of marker genes. PCR using primerslabeled a and b in A, lanes 1-2: two independent bacmid recombinantsfollowing ET recombination, lanes 3-6: four independent recombinantsfollowing Cre mediated recombination to remove the bacterial selectablemarkers, lane 7: no template, lane 8: unmodified bacmid DNA template,lane 9: plasmid DNA template containing the selectable marker cassette,lane 10: DNA marker. PCR products corresponding to the sizes predictedfor the parental and recombinant products of the Cre mediatedrecombination are labeled P and R respectively.

FIG. 2C shows a sequencing trace file of a representative recombinantfrom the PCR analysis in B confirming the presence of a defective loxPincorporating the loxP71 and loxP66 arms that render the recombinantincapable of undergoing further Cre-mediated recombination (sequencesshown in SEQ ID NOS: 1 and 2).

FIG. 2D is a bar graph showing renilla luciferase activity of theparental and recombinant bacmids from B when transfected into insectcells. Renilla luciferase activity was assayed at 48 hours postinfection after passage of the recombinant virus. Background activityfrom uninfected cells is labeled Cells.

FIG. 3A shows identification of additional sites for expression ofrecombinant proteins in the AcMNPV genome. Cartoon of AcMNPV showingrelative positions of loci used for protein expression.

FIG. 3B is atable showing loci used for insertion and any additionalchanges made to locus.

FIG. 3C is a bar graph showing relative Renilla luciferase activity at48 hours post infection with virus modified to contain an additionalfirefly luciferase expression cassette at each locus indicated. Allviruses had the same Renilla luciferase expression cassette at the p10locus under control of the p35 promoter. Error bars indicted thestandard deviation of five replicates for each locus.

FIG. 3D is a bar graph of normalised firefly luciferase activity showingrelative expression of a polyhedrin promoter-firefly luciferasepolyhedrin terminator cassette inserted at each locus as indicated.Error bars indicate the standard deviation from 5 replicates for eachlocus. Firefly luciferase was normalised using the Renilla luciferasecontrol expressed from the same genome. Firefly luciferase insertions atthe orf11, v-fgf, pe and orf23 loci were excluded due to Renillaluciferase levels more than 2 logs lower than control virus. Virus Phhas firefly luciferase at the polyhedrin locus and Renilla luciferase atthe p10 locus. Virus Ph* has the same p10 Renilla luciferase insertionbut no firefly luciferase insertion.

FIG. 4 shows expression of influenza M1 from egt and polyhedrin loci.Left panel shows Coomassie stained SDS PAGE gel of total protein fromcells infected with control baculoviruses (lanes 1 and 4), egt-M1 (lane2) and YM1-M1 (lane 3). The sizes of protein markers (lane M) areindicated on the left hand side of the gel. Right panel shows Westernblot of duplicate gel probed with anti-H7N7 antiserum.

FIG. 5A shows co-expression of influenza M1 and HA and formation ofVLPs. Left panel shows Coomassie stained gel of total protein; rightpanel shows western blot of duplicate gel probed with anti-H7N7influenza virus serum. Cells were infected with dual baculovirusexpressing HA and M1 (lane 1), baculovirus expressing HA only (lane 2),cells control (lane 3), baculovirus expressing M1 only (lane 4).

FIG. 5B shows negative stain EM images of influenza VLPs.

FIG. 5C shows negative stain EM images of immunogold (HA) labelledinfluenza VLPs with gold particles arrowed.

FIG. 6A shows production of baculovirus co-expressing 4 proteins. Celllysate from cells infected with baculovirus expressing VP2 (lane 1), VP5(lane 2), VP3 (lane 3) and VP7 (lane 4), uninfected cells (lane 5) orall 4 proteins (lanes 6-10), position of marker proteins (M) and size inkDa is as indicated.

FIG. 6B shows production of baculovirus co-expressing 4 proteins. Celllysate for cells expressing VP5, VP2, VP3, VP7 (lanes 1-4 and partiallypurified VLPs (lane 5).

FIG. 6C is a table showing locus of insertion and viral promoter usedfor expression of each BTV protein.

FIG. 6D shows immunogold negative stain EM (for VP5) showing purifiedBTV VLPs.

FIG. 7 shows crystalline plates formed on overexpression of CCT5.Quadrilateral protein aggregates in culture media of very lateinfections with baculovirus expressing CCT5.

FIG. 8A shows expression of CCT in insect cells. Coomassie stained celllysate from cells infected with baculovirus expressing CCT1-CCT8 (lanes1-8, respectively), cells only (lane 9) or baculovirus coexpressing 7CCT subunits (lane 10).

FIG. 8B shows a Western blot of duplicate gel using polyclonalanti-mouse CCT antiserum.

FIG. 8C includes tables showing different genetic loci and promotersused to express each of the CCT subunits.

EXAMPLES

The baculovirus expression system has well established potential for theproduction of large amounts of correctly folded eukaryotic proteins forenzymatic and structural studies. However, it is becoming increasinglyclear that many, if not most, proteins are active within cells ascomplexes made up of the products of several distinct genes. Given theacceleration in the pace of discovery of protein structures, there is areal need to establish systems for the rapid and reliable production andpurification of protein complexes. This is particularly true of largercomplexes that require the simultaneous expression, and eukaryoticfolding and processing, of many protein subunits. Previously theinventors have developed baculovirus transfer vectors capable ofexpressing 2, 3, 4 and 5 proteins from the same baculovirus genome. Theinventors' main goal in the previous studies was to produce a systemcapable of synthesizing large amounts of viral protein complexescontaining non-equimolar amounts of virus encoded proteins forstructural and enzymatic studies. One of the observations fromexperience with these systems is that single baculoviruses expressingmultiple genes are much more efficient at forming the desired proteincomplexes than co-infection of insect cells with multiple baculovirusesexpressing single genes. In the current study the inventors have usednew technologies to exploit this observation for the production ofrecombinant baculoviruses expressing multiple proteins for biologicallyrelevant mammalian protein complexes.

In particular, the inventors have adapted and improved new bacterialchromosome engineering technologies for the rapid and efficientproduction of recombinant baculoviruses expressing multiple proteinssimultaneously. The new systems developed are 30 times more efficientthan conventional processes and allow the routine insertion of genes atany locus within the baculovirus genome. These studies have allowedidentification of 7 new genetic loci within the baculovirus genome thatallow high level expression of recombinant protein. Furthermore, theinventors have demonstrated that multiple iterative rounds ofrecombination can be carried out allowing the generation of virusgenomes expressing multiple proteins in a complex from separate singlegene insertions. As examples, the inventors have expressed VLP proteincomplexes for influenza A (H7 subtype) and bluetongue virus (serotype1), as well as the 8 subunit mammalian chaperone complex CCT (TCP), thatis a focus for cancer studies.

Materials & Methods

Multi-locus single gene insertions are achieved in E. coli using lambdared recombination. The invention includes the use of baculovirus locithat have not previously been used for multi-protein expression, and theincorporation of selectable markers that can be removed from thebaculovirus genome and subsequently reused. Unlike other systems, therecombinant protein genes are not flanked by repeat sequences, improvingtheir genetic stability.

The transfer vectors contain a region of AcMNPV to target homologousrecombination, an expression cassette (AcMNPV promoter, polylinker,polyadenylation signal) and a bacterial selection cassette. The AcMNPVpromoter used is from either viral late (e.g. p35) or very late (e.g.Polyhedrin, p10) genes. The bacterial selection cassette consists ofmutant LoxP site—bacterial selectable marker—mutant Lox P site. Themutant Lox P sites are variants on the LoxP66 and LoxP71 variants (9).The LoxP sites for each selectable marker are designed such that onrecombination the marker is deleted destroying the LoxP site thatremains. Each selectable marker has different LoxP mutant arms so thatrecombination between different selectable marker genes inserted in thesame baculovirus will not occur.

Choice of System used for Multi-Locus Expression of Recombinant Proteins

The original research plan involved the use of homologous recombinationin insect cells, or the alternative method of ET recombination in E.coli, to allow efficient insertion of expression cassettes forrecombinant proteins at different loci within the baculovirus genome.Our preliminary data had suggested that linearization of transfer vectorresulted in a significant increase in the recombination frequency (up to˜30%) at a locus (p10) that did not have positive selection other thanthe recombinant protein produced. At the start of the research projectthe two systems (ET recombination and insect cell recombination) wereconsidered with regards to their reproducibility and time taken tocomplete insertion of a recombinant gene in a particular locus and checkexpression of the resulting protein. In terms of time taken for eachround of expression the ET recombination system was faster, mainlybecause of reduced time needed to prepare the baculovirus genome for theinsertion of genes at a second genetic locus in the baculovirus. Inaddition it was possible to design an approach that allowed confirmationof genetic insertions independently of transgene expression, andtherefore the need to grow virus in insect cells to check expression wasless critical than for the wholly insect cell based system. Secondly,although it is possible to co-introduce a marker with inserted genes inthe baculovirus system, the number of such markers is limited and eachcould only be used once. Since one of the goals of the project was toexpress the mouse CCT (TCP1) complex (8 subunits) the ET recombinationapproach was adopted as the research priority at an early stage as thissystem was faster and more able to accommodate multiple geneticinsertions.

Example 1

Development of Reagents Allowing Efficient Selection of ET Recombinants

Initial experiments using the ET approach were disappointing.Experiments were performed in which a reporter gene (luciferase) wasinserted into a bacterial artificial chromosome carrying the full AcMNPVgenome (Bacmid) and selected for by a chloramphenicol resistance genethat was co-integrated with the luciferase reporter. Althoughchloramphenicol resistant bacterial colonies were recovered, subsequentanalysis revealed that they did not contain the correctly modifiedBacmid with the luciferase reporter. Similar results were obtained whenthe reporter, which was designed to only be expressed in insect cells inthe presence of replicating AcMNPV, was changed to GFP. However, whenthe GFP construct was used for recombination in E. coli, withoutchloramphenicol selection, and Bacmid DNA prepared for the wholetransformed E. coli population, then this DNA resulted in a fewfluorescent foci when transfected into insect cells (Sf21). These datasuggested that the problem was not with the recombination itself, butwith the post-recombination selection of bacteria containing theinsertion. To overcome these problems the inventors designed a newselection cassette that incorporated a bipartite marker system based onLacZα fragment and Zeocin resistance gene flanked by modified LoxPrecombination sites (FIG. 1A). Using this system enough Zeocin was addedto the selection plates to reduce but not eliminate background colonygrowth and recombinants were selected based on blue colony phenotype inthe presence of IPTG and X-gal. To assess the relative efficiency ofrecovering recombinants using the chloramphenicol (cat) and bipartiteselection systems the inventors carried out an experiment whererecombination competent E. coli containing unmodified Bacmid wereelectroporated with 30 ng (˜12.5 fmol) of either the chloramphenicolbased or bipartite selection cassette (FIG. 1B). As an ideal system forincorporation of foreign protein coding sequences would not involverepeated PCR of the inserted gene the inventors also compared theefficiency of recombination between the PCR amplified DNA, that is theroutine for ET recombination, and restriction enzyme released, gelpurified, DNA. PCR primers were designed such that the ends of the PCRproducts corresponded to the ends of the restriction enzyme releasedfragments.

The bipartite selection resulted in a 20 fold increase in the number ofpositive colonies compared to the chloramphenicol only selection whenthe DNA fragment recombining was generated by PCR and a 30 fold increasewhen released by restriction enzyme digestion of plasmid DNA (FIG. 1B).These differences were significant (t-test, p=0.03). For thechloramphenicol selection there was no difference between the number ofcolonies recovered by PCR and restriction enzyme generated DNA. However,for the bipartite selection the mean number of colonies was four timeshigher for restriction enzyme released DNA compared to PCR amplified DNA(t-test, p=0.05). To confirm that recombinants from the new bipartiteselection were genuine, representing modified bacmid containing theinserted expression construct, PCR was carried out on bacmid DNApurified from positive colonies. One primer was designed to a sequenceinside the Zeocin resistance marker and one primer targeted a sequencepresent only in the bacmid DNA flanking the correct insertion site andnot in the transfer DNA. Thus PCR product would only be produced whererecombination had occurred between the linear DNA used for recombinationand the bacmid. 12 separate bacmid DNA samples were tested using thismethod (FIG. 1C, lanes 1-12), all were positive for the PCR productdiagnostic for correctly targeted recombination. In contrast, neitherbacmid DNA alone, nor plasmid DNA containing the DNA fragment used forrecombination was able to act as template to produce the PCR product(FIG. 1C, lanes 14 and 15, respectively). To further confirm that therecombination had resulted in the recovery of infectious recombinantbaculovirus the same 12 PCR positive bacmid clones were transfected intoSf21 cells and passaged twice, then Renilla luciferase activity in cellsinfected with each of the recombinants was assayed at 48 hours postinfection. Cells infected with each of the 12 recombinant viruses hadRenilla luciferase activities that were 106 fold above background (FIG.1D). Based on these data the inventors proceeded to use the bipartiteselection for further investigations.

Example 2

Cre Mediated Removal of the Selectable Marker Allows Multiple Rounds ofRecombination with the Same Bipartite Selection

In order to express protein complexes with multiple different subunitsusing single locus insertions, the bipartite marker system was designedsuch that the selection cassette was flanked by modified LoxP sites.These sites incorporate both the lox66 and lox71 mutations that limitCre mediated recombination to a single round (1) and a mutation in thespacer reducing homology to wildtype loxP sites. Thus, incubation ofmodified bacmid with Cre recombinase results in the removal of thebipartite selectable marker and inactivation of the lox recombinationsite but leaves behind the baculovirus expression cassette (FIG. 2A). Toconfirm that this strategy could be used successfully to engineermultiple insertions in the bacmid DNA, Cre recombination was used toremove the bipartite marker from bacmid in which the Renilla luciferasereporter had been inserted. Recombination was achieved in E. coli usingthe EL350 cell line (2), which has both an integrated lambda prophageexpressing exo, bet, and gam under control of the temperature regulatedλPL promoter and Cre recombinase under control of an arabinose induciblepromoter. EL350 cells containing three of the bacmids modified toincorporate the Renilla luciferase-bipartite selection insert (FIG. 1)were induced with arabinose then spread on plates containing kanamycin,to select for the bacmid, and X-gal, to screen for colonies that hadlost the selectable marker by Cre mediated recombination. Bacmid DNA waspurified from four putative recombinants and Cre mediated recombinationconfirmed by PCR using primers flanking the selectable markers (FIG.2B). All four recombinants had PCR products consistent with the sizeexpected from successful Cre recombination. This was further confirmedby sequencing across the modified loxP site of the 4 recombinants (FIG.2C). The recombinants contained a disabled loxP site that incorporatesboth the loxP71 and loxP66 mutations and is not competent for furtherrounds of recombination. To further confirm that Cre mediated bacmidrecombinants remained viable in insect cells, bacmid DNA was transfectedinto insect cells and luciferase activity assayed after two passages asbefore. All recombinants had Renilla luciferase activity that wasequivalent to the parental bacmids before Cre recombination (FIG. 2D).

Example 3

Identification of Genetic Loci in the Baculovirus Genome Suitable forHigh Level Expression of Heterologous Proteins

Despite the extensive protein expression work that has been undertakenin the baculovirus expression system most expression has focused onreplacement of the polyhedrin or p10 genes to express recombinantproteins. Relatively little literature describes the use of alternativeloci for the expression of recombinant protein. In order to test whetherselection could be used efficiently at different baculovirus geneticloci, a second round of recombination was carried out on one of thebacmids already containing the Renilla luciferase gene. In theseexperiments, the same polyhedrin promoter-Firefly luciferase-polyhdrinterminator was inserted independently at a total of 13 different geneticloci (ctx, orf11, egt, orf23, v-fgf, 39k, orf51, gp37, iap2, chiA, pe,odv-e18 and odv-e56) generating dual expression baculoviruses forRenilla and firefly luciferase proteins (FIG. 3A). Loci were selected assites for insertion in these experiments by determining whichbaculovirus genes are non-essential for virus growth in tissue culture,and whether the arrangement of baculovirus genes at particular locifavoured insertion of an additional expression cassette. Nevertheless,some additional changes were made at some loci, particularly changesthat resulted in knock-out of particular baculovirus genes (FIG. 3B).Recombinant viruses were passaged twice in Sf21 insect cells and on thethird passage cells were harvested at 48 hours post infection, lysed,and assayed for both firefly and Renilla luciferase activities. Renillaluciferase activity was used as a marker for virus replication andprotein expression as all the recombinants had the same p35 promoterdriven Renilla luciferase cassette in the p10 locus. Expression offirefly luciferase at each new locus was compared to a virus carryingfirefly luciferase gene at the polyhedrin locus and the same Renillaluciferase reference gene. Of the 13 loci tested, 9 had Renillaluciferase activities which were at least 10⁵ fold above background andof these 8 (ctx, egt, orf51, gp37, iap2, chiA, odv-e18 and odv-e56) hadRenilla luciferase activity which was at or above the activity measuredfor the parental Renilla luciferase only, and polyhedrin locus fireflyluciferase controls (FIG. 3C). The four loci (orf11, v-fgf, pe andorf23) which resulted in virus which gave Renilla luciferase activitythat was within 10 fold of the background activity were excluded fromfurther analysis. For the remaining viruses, the Renilla luciferaseactivity was used as a reference to normalise firefly luciferaseactivity and obtain a measure of relative expression of the fireflyluciferase from each locus (FIG. 3D). 7 loci (ctx, egt, 39k, orf51,gp37, iap2 and odv-e56) had firefly luciferase activity which was atleast 10⁶ fold higher than background and similar to that when the samegene was expressed from the polyhedrin locus (FIG. 3D). Two viruses(chiA and odv-e18) had high levels of Renilla luciferase but relativelypoor expression of firefly luciferase.

This study reveals that high level expression of foreign proteins ispossible from several genetic loci within the baculovirus genome andidentifies seven loci (ctx, egt, 39k, orf51, gp37, iap2 and odv-e56), inaddition to polyhedrin and p10 that give good expression. Of these sites39k, orf51 and gp37 are insertions into the DNA flanking the codingregion of the gene and do not directly interrupt protein expression. Incontrast, insertions into the ctx, egt, iap2 and odv-e56 each would beexpected to prevent expression of the corresponding proteins from thesegenes. The first three of these genes have previously been described asnon-essential to the growth of the virus in cell culture (3-6).Truncation of the ODV-E56 protein has also been reported (7).

Of the loci that did not give good expression of the firefly luciferasereporter, four (orf11, v-fgf, pe and orf23) also resulted in a reducedexpression of Renilla luciferase marker protein which was present in allrecombinants. As the focus of the study was to identify sites which weresuitable for insertion of very late promoter expression constructs theprecise reason for this reduced expression was not investigated.Possibilities include locus-specific effects on virus replication ortranscription, and disruption of essential promoter or enhancer elementsfor flanking genes. The low level expression of firefly luciferase inthe chiA and odv-e18 insertion viruses was unexpected for differentreasons. Other reports have recorded insertion of recombinant proteinexpression cassettes into the chiA locus (8, 9). It is possible that thereduced level of expression seen with the firefly luciferase gene inthis locus in our experiments is due to the effects on genes flankingthe insertion. For odv-e18, recent reports using the same mutant virushave suggested that this protein is essential for budded virusproduction and cell to cell movement (10, 11). These studies were basedon mutants in which there was a deletion within the coding sequence ofodv-e18 and the upstream flanking gene. In the experiments, whereodv-e18 was inactivated by mutation of the ATG of the coding sequence toGAT followed by insertion of the firefly luciferase cassette at thispoint in the gene it was possible to recover infectious virus.Expression of Renilla luciferase on the third passage equivalent to thatin the parental virus suggests that there was no impairment of theability of this mutant virus to replicate. However, given the ˜2 logreduction in firefly luciferase levels compared to virus without thismutation it is not possible to rule out the possibility that a smallpopulation of virus in which the mutation was repaired was complementinga second population expressing the reporter gene.

Example 4

Expression of Protein Complexes Using Multi-Locus Baculovirus Expression

Example 3 focused on the use of reporter genes to quantitatively assessthe potential of different baculovirus loci to express recombinantprotein. To establish whether it is possible to express and recoverrecombinant protein complexes, three specific complexes were targetedwith different numbers of protein subunits. Virus-like particles wereproduced for Influenza (A/seal/Mass/1/80) H7 subtype by co-expression ofthe viral M1 and IIA proteins (2 protein complex), and for Bluetonguevirus (BTV) serotype 1 by co-expression of VP2, VP3, VP5 and VP7 (4protein complex). In addition, the inventors targeted expression of all8 subunits of the mouse CCT chaperone complex to be used for furtherfunctional and structural studies.

Influenza A VLPs

For these experiments all influenza genes were from the SC35M mouseadapted strain derived from the H7N7 (A/seal/Mass/1/80) isolate,obtained from researchers at Philipps University Marburg, Germany

To test whether the multi-locus expression system was able to produceprotein complexes with two proteins, the coding sequence for theinfluenza A M1 protein was initially inserted into the egt locus underthe control of the polyhedrin promoter. For comparison, the same genewas inserted into a conventional baculovirus expression vector (pAc-YM1)targeting the polyhedrin locus. Following recombination, both resultingbaculoviruses expressed the M1 protein. Levels of expression of theprotein from the egt and polyhedrin loci were both significantly higherthan for any other viral or cellular protein in baculovirus infectedcells when assessed by coomassie stained SDS-PAGE (FIG. 4).

To generate a dual virus expressing both M1 and IIA, the bacterialselection cassette was removed from the M1 expressing Bacmid by Crerecombination and the HA gene from the same influenza strain(A/seal/Mass/1/80) inserted at the p10 locus by a second round of ETrecombination. Co-expression of M1 and HA from the resulting virus wereconfirmed by SDS-PAGE and western blot analysis as before (FIG. 5A). Inaddition, influenza VLPs were isolated from the culture medium ofinfected cells, purified by density gradient ultracentrifugation andvisualised by negative stain EM analysis (FIG. 5B). To ensure that VLPsvisualised were indeed influenza, particles were immunogold labeledusing antibodies specific for influenza HA (FIG. 5C).

Taken together, these data were good evidence that the multilocusapproach could be used successfully for recombinant protein expressionand that it was practical to produce such a recombinant virus throughtwo rounds of insertion of the bipartite selectable marker system. FluVLP have been widely tested by others and shown to be immunogenic inmice and ferrets. This method of constructing the VLP has the advantagethat it is possible to have the baculovirus genome with M1 alreadypre-integrated and ready for expression. Thus, to make VLP it shouldonly be necessary to perform a single round of recombination to add theHA gene from whatever emerging flu subtype is required for vaccineproduction. For VLP based flu vaccines this would simplify the necessarycloning and potentially increase the speed with which new types of VLPcould be made.

BTV1 VLPs

Unlike influenza, where VLP can be formed by expression of just twoproteins, BTV VLP require the co-ordinated expression of four structuralproteins (VP2, VP3, VP5 and VP7). In order to further demonstrate theusefulness of the new system for recombination, viruses were producedthat expressed each protein singly and the combination of all four BTVstructural proteins (FIG. 6). One of the advantages of the new system isthat viruses expressing single proteins and multiple proteins can bemade from the same set of transfer vectors. This facilitates productionof control viruses expressing single proteins to act as markers for theposition of proteins co-expressed in the full complex. For the BTV VLP adifferent set of loci were used from those in the influenza example(FIG. 6C). In addition, the new system was combined with the orf1629knockout Bacmid described by Zhao et al., (12) to allow use of aconventional transfer vector at the polyhedrin locus to express one ofthe subunits of the complex. Formation of VLPs was confirmed by particlemorphology under negative stain EM, combined with immunogold labellingfor one of the outer capsid proteins (VP5) (FIG. 6D).

Mouse CCT

The application of the present invention is not limited to the formationof virus-like particles. Indeed, as many cellular proteins are presentin cells as complexes with more than one subunit, the efficientformation of these complexes has application to a range of fields. Todemonstrate the usefulness of the system to other studies the chaperonecomplex CCT was chosen. This complex has implications in cancer researchand with 8 subunits represents a significant challenge for proteinexpression. Indeed it would not have been possible to express thiscomplex in the baculovirus system without the vectors and methods of thepresent invention. Eight transfer vectors, each expressing one of thesubunits for the mouse CCT and each targeting a different locus,identified in Example 3 above, were constructed. These were used togenerate recombinant virus expressing each subunit alone andcombinations of subunits. For one of the subunits (CCT5) an unusualphenotype was noted when the protein was over expressed in cells.Crystalline needles were noted inside cells and, late in infection whencells had ruptured, quadrilateral plates of crystalline material werenoted in the culture medium (FIG. 7). A purification scheme wasgenerated for this protein and this was supplied with preliminarycrystallisation conditions and infected cell material to thecollaborator for the CCT part of the project.

Although expression of some of the other CCT subunits resulted invisible aggregates in cells late in infection, none resulted inaggregates with such a regular appearance.

All 8 CCT subunits had high level expression (FIG. 8A) and cross reactedwith a polyclonal antiserum raised to mouse CCT. There was some crossreaction between one of the endogenous insect cell CCT subunits and thepolyclonal antibody (FIG. 8B, lane 9). However, this endogenous signalonly co-located with 3 subunits (CCT1, CCT5 and CCT6) which allaccumulated to high level in infected cells and thus were clearlydetectable. All other subunits migrated as slightly different molecularmass proteins and thus could be distinguished from the insect cellprotein on the basis of migration.

CONCLUSIONS

For all three protein complexes there were clear differences in theaccumulation of the same recombinant protein when expressed alone or inpresence of other proteins. In almost all cases there was a reduction inthe level of protein expression when multiple proteins were expressedfrom the same virus. This was to a certain extent expected. Competitionfor proteins required for transcription, RNA processing, translation andprotein folding would predict that two highly expressed baculovirusgenes would have lower expression together than separately. However whatwas striking for both the BTV VLP and CCT examples was that reduction inprotein expression was variable between different genetic loci. Forexample with BTV, VP2, VP5, VP3 and VP7 all resulted in significant andsimilar accumulation of protein when expressed on their own. However,when combined in a single baculovirus expressing all four proteins, VP2and VP3 accumulated to lower levels than VP5 and VP7 (FIG. 6A). Thiseffect cannot be explained by the position effect of genomic integrationsince in both the single and quadruple viruses the genes were expressedfrom the same genetic loci. One possible explanation would be that theeffect was due to the different promoters that were used for thedifferent genes. Both VP5 and VP7 were expressed under control of thep10 promoter and VP2 and VP3 were under control of the polyhedrinpromoter. This would be consistent with a report in the literature wheredeletion of the p10 gene resulted in increased expression frompolyhedrin locus 13. Thus duplication of the polyhedrin promoter in thepresence of two p10 promoters would be predicted to selectively reduceexpression from the polyhedrin promoter driven genes. However, thiscannot be a complete explanation for the effect observed in the case ofthe CCT complex. Both CCT5 and CCT2 were expressed from the p10 promoterand gave high steady-state levels of recombinant protein when expressedalone (FIGS. 8A and B, lanes 2 and 5). However, when combined in thesame virus, expression of CCT5 was substantially reduced compared toCCT2 (FIGS. 8A and B, lane 10). From these data, other cis actingsequences present at the p10 locus may be contributing to the enhancedrelative expression of CCT2 and BTV VP5, which were both inserted atthis locus. The relatively low apparent expression of influenza HA wheninserted at the same site may be more related to turnover of thisprotein in the ER rather than transcriptional effects. Both CCT2 and VP5accumulate in the cytoplasm.

The inventors have improved the efficiency of the ET recombinationsystem as applied to baculovirus to the point that it can be used forroutine insertion of expression cassettes for recombinant proteins.Furthermore, they have identified seven genetic loci (ctx, egt, 39k,orf51, gp37, iap2 and odv-e56) that can be used for high levelexpression of protein and demonstrated that multi-protein complexes canbe assembled using this system using three examples (influenza A and BTVVLPs, and CCT complex).

All documents cited above are incorporated herein by reference.

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The invention claimed is:
 1. A suite of transfer vectors for inserting aplurality of genes that encode a plurality of proteins that are subunitsof a protein complex into a baculovirus sequence comprising: i) aplurality of transfer vectors, wherein each transfer vector comprises anexpression cassette comprising a eukaryotic promoter operably linked toat least one of the said genes; and ii) sequences flanking both sides ofthe expression cassette, wherein the sequences substantially correspondto sequences within a selected one of the genetic locus within thebaculovirus selected from: ctx, egt, 39k, orf51, gp37, iap2 and odv-e56and wherein different transfer vectors have different said flankingsequences.
 2. A method for producing a recombinant bacmid, comprising:bringing a bacmid and the suite of transfer vectors of claim 1 togetherto allow homologous recombination; and selecting for a recombinantbacmid that comprises an expression cassette.
 3. A method for producinga recombinant baculovirus comprising producing a recombinant bacmid bythe method of claim 2 and culturing a eukaryotic cell containing thebacmid so that a baculovirus is produced.
 4. The suite of transfervectors of claim 1, wherein the plurality of transfer vectors arebacmids.
 5. The suite of transfer vectors of claim 1, wherein theplurality of proteins interact to form a protein complex.
 6. The suiteof transfer vectors according to claim 5, wherein the protein complex isa virus like particle, or a chaperone complex.
 7. The suite of transfervectors of claim 1, wherein the plurality of transfer vectors comprise arecombinant baculovirus.
 8. A cell in culture containing the suite oftransfer vectors of claim
 1. 9. A method for producing one or moreproteins comprising culturing the suite of transfer vectors of claim 1.